This proposal concerns the mechanism of initiation of translation in eukaryotic cells. Messenger RNAs with specially contrived leader sequences will be translated in vitro to gain insight into how the small (40S) ribosomal subunit engages the mRNA, advances across the 5' noncoding sequence, and selects the AUG initiator codon. The first Specific Aim is to investigate the control of translation by mRNA secondary structure. In light of past studies which showed that translation is inhibited by a perfectly base-paired -60 kcal hairpin but not by a -30 kcal hairpin positioned between the m7G cap and the AUG codon, the effect of two -30 kcal base-paired structures placed in succession will be investigated. These experiments may shed light on the processivity of the scanning phase of initiation. Other experiments will test the hypothesis that a hairpin structure positioned exactly at the 5' end might be able to lead the mRNA onto the ribosome and thus to facilitate translation. This would contrast with the usual inhibitory effects of 5' secondary structure. The second Specific Aim is to investigate some other aspects of the scanning mechanism of initiation. Some evidence suggests that the usual preference for initiating at the first AUG codon might break down when a second AUG occurs nearby. Two hypotheses to explain this breakdown of the first-AUG rule will be tested. Following up past studies which mapped (only) the boundaries of ribosome-binding sites on mRNAs, chemical footprinting techniques will now be used to reveal internal details such as whether a purine in position -3 (which strongly favors recognition of the AUG codon) shows a special interaction, and whether the footprint shows evidence of periodicity, and whether edeine alters the footprint. Other experiments will test the hypothesis that slowing of the scanning 40S ribosome might facilitate recognition of the AUG codon. The third Specific Aim is to investigate alternative mechanisms of initiation that might operate with the long, 5' leader sequence of encephalomyocarditis virus (EMCV). A cloned 5' fragment of EMCV mRNA will be circularized as a test of the "internal initiation" hypothesis. An alternative hypothesis is that ribosomes reach the internal start site in EMCV mRNA by scanning and repeatedly reinitiating within the long 5' leader sequence. To test this idea, the coding sequence for a small peptide that inhibits translation in cis will be introduced at various positions near the 5' end of EMCV mRNA.